Review
This Review is part of a thematic series on Genetics of Cardiovascular Development, which includes the following
articles:
Transcriptional Regulation of Vertebrate Cardiac Morphogenesis
Cardiac Septation: A Late Contribution of the Embryonic Primary Myocardium to Heart Morphogenesis
Early Signals in Cardiac Development
Left/Right Patterning
Development of Specialized Cells Within the Heart
Development Gone Awry: Congenital Heart Disease
Christine E. Seidman, Guest Editor
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Cardiac Septation
A Late Contribution of the Embryonic Primary Myocardium
to Heart Morphogenesis
Wouter H. Lamers, Antoon F.M. Moorman
Abstract—Heart morphogenesis comprises 2 major consecutive steps, viz. chamber formation followed by septation.
Septation is the remodeling of the heart from a single-channel peristaltic pump to a dual-channel, synchronously
contracting device with 1-way valves. In the human heart, septation occurs between 4 and 7 weeks of development.
Cardiac looping and chamber formation bring the contributing structures into position to engage in septation.
Cardiomyocytes that participate in chamber formation do not materially contribute to septation. The (re)discovery of the
role of extracardiac mesenchymal tissue in atrioventricular septation, the appreciation that the formation of the right
atrioventricular connection is more than a mere rightward expansion of the atrioventricular canal, the awareness that
myocardium originating from the so-called anterior heart field regresses after its function as outflow-tract sphincter
ceases, and the recent finding that the myocardialized proximal portion of the outflow-tract septum becomes the
supraventricular crest have all significantly enhanced our understanding of the morphogenetic processes that contribute
to septation. The bifurcation of the ventricular conduction system is the landmark that separates the contribution of the
atrioventricular cushions and the outflow-tract ridges to septation and that divides the muscular ventricular septum in
inlet, trabecular, and outlet portions. (Circ Res. 2002;91:93-103.)
Key Words: primary myocardium 䡲 ballooning heart model 䡲 septation 䡲 fate map
T
he remodeling of the heart from a single-channel peristaltic pump into a dual-channel, synchronously contracting 4-chamber pump with 1-way valves has been studied for
well over a century now. Cardiac looping and chamber
formation (for recent reviews, see Männer1 and Christoffels et
al1a) bring the contributing embryonic heart structures into
position to engage in septation. Cardiac septation is here
considered to involve the closure of direct communications
between left and right atria, ventricles and subarterial chan-
nels, and the development of the right atrioventricular junction and left ventriculoarterial junction. In the human heart,
septation occurs between 4 and 7 weeks of development.
The ballooning heart model (Figure 1) summarizes the
results of cardiac looping and chamber formation of the
embryonic heart when it is about to engage in septation. In
this model, the atrial and ventricular chambers are situated
along the outer curvature of the heart2,3 and are more
advanced with respect to cardiomyocyte differentiation than
Original received April 10, 2002; revision received June 11, 2002; accepted June 13, 2002.
From the Department of Anatomy and Embryology, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands.
Correspondence to W.H. Lamers MD, PhD, Dept of Anatomy & Embryology, Academic Medical Center, University of Amsterdam, Meibergdreef 15,
1105 AZ, Amsterdam, The Netherlands. E-mail [email protected] or [email protected]
© 2002 American Heart Association, Inc.
Circulation Research is available at http://www.circresaha.org
DOI: 10.1161/01.RES.0000027135.63141.89
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Figure 1. Schematic drawings of the
heart just before septation (modified
from Moorman and Christoffels46). A,
4-chamber view of the embryonic heart
just before septation. Outflow tract is
deviated rightward to visualize the atrioventricular canal. Atrial appendages
(blue) and the embryonic left and right
ventricles (red) have formed as “balloons” on the outer curvature of the
heart tube, but their flanking structures
(the body of the atrium, the atrioventricular canal, the “primary ring” between
both ventricles, and the outflow tract)
retain the tubular shape and functional
properties of the primary myocardium
(purple). Transverse sections through the
atrioventricular canal and outflow tract
show the position of the endocardial
cushions and ridges. B, Sagittal section
through the primary atrial septum, the
atrioventricular canal, and the left ventricle (following the dashed line in A) shows the relation between the atrioventricular cushions and
the atrial spine. 1 indicates inferior (caudal) atrioventricular cushion; 2, superior (cranial) atrioventricular cushion; 3, parietal outflow-tract
ridge; 4, septal outflow-tract ridge; AS, atrial spine with spur (5) on leading edge of primary atrial septum (PAS); 6, body of atrium; 7,
primary ring; PIF, primary interatrial foramen; RA, right appendage; LA, left appendage; AVC, atrioventricular canal; LV, embryonic left
ventricle; RV, embryonic right ventricle; and OFT, outflow tract.
the remaining regions. These latter regions, that is, the
structures of the atrial midline, the atrioventricular junction,
the inner curvature of the heart loop, and the outflow tract,
temporally retain the properties of the primary myocardium
of the embryonic heart tube.1a Intriguingly, the morphogenetic events that lead to cardiac septation are largely confined
to these less advanced structures, implying that myocytes that
participate in the first step (chamber formation), do not
materially contribute to the second step (septation).
Description cannot provide conclusive evidence in favor of
mechanistic hypotheses. Morphological observations, nevertheless, often initiate such hypotheses. Indeed, flawed descriptions or interpretations of morphology have generated
useless hypotheses and, hence, hindered progress. Cardiac
embryology has certainly been a case in point.4 Fortunately,
the implementation of new techniques has facilitated interpretation and, hence, changed the appreciation for embryology. In this respect, in vivo labeling of cells is certainly the
most definitive technique for establishing the origin and fate
of major components of the heart (summarized in De la Cruz
and Markwald5). Unfortunately, the accuracy of this technique has its limitations, in particular when the label cannot
be positioned on an unambiguous landmark. In addition, its
application to later development, including much of septation, is often limited by poor accessibility of the relevant
structures. Despite their inherent shortcomings, phenotypic
markers have, therefore, proven to be very useful for delineating structures that participate in septation and for tracking
their fate in consecutive stages of development. Furthermore,
the morphogenetic models that were deduced from these data
can now often be tested for compatibility with extant congenital malformations, including those resulting from genetically modified mice. In the following, we will give an
account of our understanding of cardiac septation as it has
developed in our group and elsewhere over the last 10 years.
The account will focus on the mammalian heart, with an
emphasis on the developing human heart and on structures
that are considered to be prime movers in the morphogenetic
processes underlying septation. Carnegie stages6 are assigned
to all pertinent events, which may facilitate future reading,
because ultrasound data may change the longstanding relation
between developmental age and Carnegie stages,7 and should
facilitate the comparison with mouse or rat development.8
Cranial, caudal, dorsal, and ventral will be used as indicators
of orientation, with the apex of the heart always pointing
ventrally. It should be noted that, in the postnatal human
heart, these terms correspond to superior, inferior, posterior,
and anterior, respectively. For description of the atrioventricular endocardial cushions, we have retained the commonly
used terms “superior” and “inferior.”
Septation of the Atria
Left and right appendages develop from the caudal part of the
heart-forming regions. This part of the heart-forming regions
does not participate in looping but, instead, retains its left and
right identity.9 Indeed, sidedness, as revealed by the different
morphology of left and right appendages, is a determining
factor for atrial development.10 –12
The left and right atria develop symmetrically up to the
11th stage (⬇22 days9,12a), when their caudal continuation
into both sinus horns begins to move rightwards, coincident
with rapid growth of the atria and the right sinus horn,
whereas growth of the left sinus horn falls behind.6,9 The
sinuatrial junction as such becomes identifiable as the rightsided sinuatrial foramen during the 12th stage (⬇26
days).6,13,14 The protrusion of the sinuatrial foramen into the
lumen of the atrium generates the so-called venous valves.
The lower, caudal portion of the venous valves is a true fold,
whereas the upper, cranial portion is a single-layered structure, suggesting development by proliferation.15 The upper
commissure of the venous valves continues as the septum
spurium in the roof of the right atrium, whereas the ventricular ends of the valves insert near the midline on the so-called
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Figure 2. Sidedness of the caudal portion of the preseptational heart. Serial transverse sections through the caudal part of the atrioventricular canal of a stage 15 heart (35 days). Note the extension of the myocardium (A, stained for the presence of ␣-myosin heavy
chain), the distribution of vessels in this area, including the already prominent pulmonary vein (B, stained for the presence of endothelium), and prominent expression of the left-sided marker CK-B (C) in the epithelium of the pleural cavity overlying left sinus horn and the
left lung bud. LSH indicates left sinus horn; RSH, right sinus horn, PV, pulmonary vein; AS, atrial spine; and IEC, inferior endocardial
cushion. Bar⫽200 ␮m.
“atrial spine.” The venous valves therefore occupy a plane
that intersects the sagittal plane of the atrium at a 45° angle.
During the 13th stage (⬇28 days of development), the
pulmonary vein is seen to develop from an endothelial
evagination into the dorsal mesocardium, just cranial to the
“sinus septum” that marks the confluence of both sinus
horns.13,16 –19 Relative to the size of the heart, the dorsal
mesocardium, that is, the attachment of the caudal portion of
the heart tube to the dorsal body wall, is still a relatively large
structure at this stage12a,20 and surrounds the orifice of the
pulmonary vein as a rim that generates the typical pit-like
appearance of the orifice of the pulmonary vein when
inspected from the atrial cavity.17,18 Cranial to the pulmonary
pit, the primary atrial septum develops as a crescent-shaped
muscular septum that expands from the dorsal wall of the
atrium toward the atrioventricular canal in the 5th and 6th
weeks (14th through 16th stages; Figure 1). The left-sided rim
of the pulmonary pit is a transient structure, but the rightsided rim increases in size to become the so called “spina
vestibuli.”17,19,21–24 This structure, first described by His,21
owes its name to the Latin translation of the German word for
atrium (“Vorhof”) and should therefore be renamed the
“atrial spine” in English texts. The body of this extracardiac
tissue penetrates the atrium as a prong of mesenchyme on top
of the inferior atrioventricular endocardial cushion,17,21,24
whereas its cranial spur follows the developing primary atrial
septum as the thin mesenchymal cap on its leading
edge.15,23,24 The communication between the left and right
atrium underneath the primary atrial septum (the “primary”
foramen) closes in the second half of the 6th week (16th and
17th stages6,18,24) when the body of the atrial spine and the
mesenchymal cap on the primary atrial septum merge and
fuse with the superior endocardial cushion. Shortly thereafter,
both atrioventricular endocardial cushions begin to fuse,
creating separate left and right atrioventricular connections.25–27 Meanwhile, a number of fenestrations develop in
the dorsal portion of the primary atrial septum to form a new
interatrial communication, the “secondary” foramen.
There is some argument as to whether the pulmonary
orifice lies ab initio within the confines of the atrium13,17,28 or
is secondarily recruited from the sinus venosus.18,19 The
argument centers on the question whether or not a temporarily recognizable (during the 12th stage only) shallow fold
to the left of the pulmonary pit is an upward extension of the
caudal sinuatrial fold. This temporary ridge is argued to be a
sinus venosus structure, because it expresses HNK-1 in the
chick,29 but has disappeared when the pulmonary vein acquires access to the left atrium.29 These arguments underscore
that the initiation of pulmonary vein development is intimately linked with the development of asymmetry within the
venous pole of the heart. Indeed, the dorsal mesocardium
itself is highly polarized, because its left-sided epithelium
strongly expresses the left-sided markers creatine kinase-B
(CK-B; Figure 2)15 and Pitx-2,30 whereas its right-sided
counterpart does not. Furthermore, the myocardium surrounding the developing pulmonary vein (“pulmonary myocardium”) is Pitx2-positive,11 indicating its left identity. The
pulmonary vein is therefore a left-sided structure in the dorsal
mesocardium, lying to the left of the atrial spine and on top of
the inferior atrioventricular cushion when it enters the atrial
cavity.15,18
The present description of atrial septation differs from that
in standard textbooks in the decisive role that is attributed to
the atrial spine in septating the atrium and closing the primary
foramen. The body of the spine is tightly attached to the
inferior atrioventricular cushion, from which it can only be
distinguished immunohistochemically.15,24 There is still some
argument whether the mesenchymal cap on the leading edge
of the primary atrial septum is of extracardiac origin, as we
claim based on its staining properties,15,24 or arises from a
local epitheliomesenchymal transformation similar to that
seen for the atrioventricular cushions.31–33 Although the
extent to which the spine penetrates the atrium is therefore
still being disputed, there is general agreement that the
expansion of the body of the spine over the endocardial
cushions is instrumental for the closure of the primary
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interatrial foramen. The importance of the atrial spine for
atrioventricular septation is also supported by data from
mutant mouse embryos in which defective development of
the atrial spine, including its spur, is associated with persistence of the foramen primum and the development of atrioventricular septal defects.34,35 In these mice, the atrioventricular cushions almost always fuse.
Soon after closure of the primary foramen, the spine
becomes muscular.24,33,36 Myocardialization starts near the
sites of attachment of the primary atrial septum and
the venous valves on the spine. In preseptation hearts, the
sinuatrial fold and the pulmonary pit are adjacent structures in
the caudal wall of the atrium.17,18 When the atrial spine
expands into the atrium, the venous valves become anchored
to the spine. Myocardium largely replaces the mesenchymal
component of the spine, except in its center, where Todaro’s
tendon forms.24,33 The primary atrial septum forms the flap
valve of the oval foramen in the formed fetal heart, whereas
the myocardialized atrial spine forms the ventral and caudal
rims of the oval fossa. The dorsal and cranial rims of the oval
fossa (the “limbus”) are formed by the so-called secondary
septum of the atrium, a structure that develops as an infolding
of the dorsal atrial wall rather than as a septum between the
entrance of the systemic and pulmonary veins. It should be
emphasized that the structure, which develops coincident
with the regression of the left venous valve, is difficult to
identify in the embryo and only becomes a pronounced fold
in the course of the second trimester of pregnancy.37
The observation that the definitive atria appear to comprise
4 transcriptional domains, namely, the myocardium of the
sinus venosus, the atrial appendages, the atrioventricular
canal, and the dorsal mesocardium,11 suggests that these
domains reflect tissue sources and, hence, the lineage of atrial
myocardium. The domain of the dorsal mesocardium includes, in addition to the atrial spine, the components of the
definitive interatrial septum and the myocardium surrounding
the pulmonary veins. The dorsal fold that forms the secondary
atrial septum develops at the boundary of the expression of
the left-sided markers CK-B15 and Pitx2,38 and delineates the
original midline of the atria. This finding implies that the
primary atrial septum is a left-sided structure. In agreement
with this hypothesis, the primary atrial septum abundantly
expresses the left-sided markers CK-B15 and Pitx2.38
Septation of the Atrioventricular Junction
Because the atrioventricular canal and the ventricles are part
of the heart tube that undergoes rightward looping,1,38,39 the
originally left-sided portion of the heart tube acquires a
ventral position and the right-sided portion a dorsal position.
Hence, the left and right ventricles are each made up of
contributions of both the left- and right-sided heart fields.
Although this lack of a “simple” bilateral symmetry in the
atrioventricular canal and ventricles has been known for more
than 40 years,39,39a its consequences for the morphogenesis of
this region during septation have only recently been acknowledged. Another complicating development is the more pronounced growth in the dextro-caudal portion of the atrioventricular canal and the adjacent inlet portion of the right
ventricle (both derivatives of the right-sided heart field)
relative to that of the left-sided counterparts in the 5th and 6th
weeks.9,14,24,40 As a result, a generally accepted account of the
morphological changes that accompany the remodeling of the
atrioventricular junction during septation is still lacking. The
present account is largely based on the phenotypical identification of structures24,40,41 and the interpretation of the
spectrum of malformations that is seen in pertinent mutant
mice.34,35
Up to the middle of the 6th week (15th stage), the atrioventricular canal is largely positioned above the left ventricle.
Because a large “primary” interventricular foramen exists and
because the ventricle remains relaxed until atrial contraction is
completed, atrial blood can pass directly to the right ventricle.3
However, after separate atrioventricular connections have been
established, the right atrium must be in direct contact with the
right ventricle. The necessary remodeling of the right atrioventricular junction proceeds coincident with the fusion of the
atrioventricular cushions late in the 6th and in the 7th week.24 To
understand this remodeling process, one has to keep in mind that
growth in the preseptational heart is strongest in the outer
curvature and virtually absent in the inner curvature.1a,39a,42,43 As
a result, the “junctional” myocardium between atria and ventricles and that between embryonic left and right ventricles share
the myocardial wall in the short inner curvature24 (Figure 1). In
the human heart, the myocardium surrounding the atrioventricular canal is distinct from that of the atria and ventricles in that
it does not express creatine kinase M (CK-M),24,44 whereas the
myocardium surrounding the primary interventricular foramen
distinguishes itself from that of the ventricles in that it carries the
sulfoglucuronyl-carbohydrate epitope that is recognized by the
Gln2/HNK-1/Leu7 antibodies.24,45,46 Because this latter myocardium surrounds the primary interventricular foramen and is the
precursor of the ventricular conduction system, it was labeled the
“primary ring.”41 The phenotype of the portion of the myocardium in the lesser curvature that is shared by the atrioventricular
canal and the primary ring is therefore both CK-M–negative and
sulfoglucuronyl-carbohydrate–positive.
Because growth in the atrioventricular canal is more
pronounced on the atrial than on the ventricular side, and on
the dextro-caudal (that is, originally right) side than on the
opposite side, the atrioventricular canal becomes an asymmetric, funnel-shaped structure.24,40 This asymmetric growth
in the atrioventricular-canal region also comprises the part of
the inner curvature that the atrioventricular canal shares with
the primary ring, as well as the adjacent part of the right
ventricle. We previously dubbed the right-ventricular structure that forms as a result of the expansion of the atrioventricular canal within the confines of the primary ring the
“tricuspid gully.”40 A gully that is similar to, but substantially
smaller than the tricuspid gully, is present at the junction of
the atrioventricular canal with the left ventricle, again emphasizing the pronounced left-right asymmetry in growth
during septation.
At 5 weeks, a muscular septum is identifiable between the
trabecular portions of both ventricles. We define this portion
of the muscular septum as its “middle” portion. The caudal
portion of the muscular septum, that is, the portion supporting
the developing atrioventricular canal and the inferior endocardial cushion, develops later, coincident with the changing
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configuration of the atrioventricular canal, as can be demonstrated by the topography of the emerging ventricular conduction system: at 5 weeks (15th stage), the bundle branches
still occupy the caudal wall of both ventricles, whereas at 6
weeks (17th stage), these structures, including the atrioventricular bundle of His, are found on the crest of the caudal
portion of the muscular ventricular septum.24,40 Indeed, casts
of the cardiac cavities confirm the rapid development of the
caudal portion of the septum between 5.5 and 6 weeks (15th
through 17th stages).14 We define this caudal portion of the
muscular ventricular septum as its “inlet” portion.
The tricuspid gully is mounted medially on the rim of the
inlet portion of the ventricular septum. Interestingly, this
portion of the septum is bent leftward relative to its middle,
trabecular portion (Figures 3A through 3D), the bend thus
marking the boundary between both components. As argued
above, the pronounced growth of the dextro-caudal portion of
the atrioventricular canal, the tricuspid gully, and the inlet
portion of the muscular ventricular septum appear to be
linked events. Indeed, mice with atrioventricular septal defect
not only suffer from a persisting primary foramen of the
atrium and a left-sided position of the atrioventricular canal,
but also fail to develop the tricuspid gully and lack a right
ventricular inlet (Figures 3H through 3J).35 Similarly, in
human hearts with atrioventricular septal defect, not only the
rightward expansion of the atrioventricular canal, but also the
development of the inlet portion of the ventricular septum
appears to be deficient, giving the septum of such hearts a
“scooped-out” appearance.47
The tricuspid gully develops as a single layer of myocardium within the confines of the primary ring, with the right
bundle branch forming its cranial boundary.24 During and
after formation of the tricuspid gully, the right atrium has
access to the right ventricle via the relatively small cranial
exit of the gully, that is, after passing over the developing
septomarginal trabeculation containing the right bundle
branch. This portion of the right atrioventricular connection
corresponds with the prepapillary orifice of the tricuspid
valve. After the formation of the gully, the right ventricle
begins to increase in size. Coincident with this development
during the 18th stage (6 to 6.5 weeks), fenestrations appear in
the floor of the gully that will form the much larger caudal or
postpapillary orifice of the tricuspid valve.40
The morphogenetic remodeling that accompanies atrioventricular septation, that is, the formation of the right-sided
portion of the atrioventricular canal and the adjacent part of
the right ventricle, affects the position of the inferior endocardial cushion more than that of its superior counterpart.
Because the endocardial cushions continue to grow during the
septational period,48 the inferior atrioventricular cushion continues to fill the expanding atrioventricular canal. Its association with the selectively expanding dextro-caudal structures
of the atrioventricular canal explains its mainly right-sided
position relative to the muscular ventricular septum and its
apparent penetration into the right ventricle on top of the inlet
or caudal component of the muscular ventricular septum.
The atrioventricular endocardial cushions form the atrial
surface of the atrioventricular valves40,49 and, at their site of
fusion, the atrioventricular portion of the membranous sep-
Cardiac Septation
97
tum of the heart. The interventricular portion of the membranous develops much later, in the course of the last trimester of
pregnancy, after the delamination of the medial leaflet of the
tricuspid valve has reached the top of the muscular ventricular septum.50 This delamination occurs within the myocardium, so that the ventricular surface of the leaflets is covered
by coarse fibrous tissue stemming from the myocardium,
whereas their atrial surface is covered with the smooth
derivative of the atrioventricular cushions.40 The plane of
delamination suggests that the myocardium of the tricuspid
gully delaminates from that of the inlet portion of the
muscular ventricular septum. Because trabeculae are absent,
the surface of the right ventricular inlet underneath the medial
tricuspid leaflet is said to be smooth. Interestingly, the plane
of delamination of the medial tricuspid leaflet changes
smoothly into that of the right atrioventricular sulcus, most
likely because their overlying structures, the tricuspid gully
and the right-sided atrial vestibule, respectively, both owe
their development to the selectively right-sided growth of the
atrioventricular-canal region during septation.24 The deepening of the right atrioventricular sulcus is therefore also an
inherent part of cardiac septation.
This account of atrioventricular septation24 differs from
existing descriptions in identifying the originally right-sided
heart anlagen as a major contributor to growth in the
atrioventricular region and the source of tissue forming the
septating structures. Although this conclusion was already
suggested by in vivo marking experiments,5 very few studies
have detailed the growth of the atrioventricular canal myocardium during the septation period. In contrast, the embryological origin of the so-called “inlet” to the right ventricle,
that is, the portion of the right ventricle that contains the
atrioventricular valve and its tension apparatus, has long been
a hotly debated issue. Two hypotheses have been put forward
to explain the development of a connection between the right
atrium and the right ventricle that is as wide as that between
the left atrium and the left ventricle: either the caudal portion
of the muscular ventricular septum grows toward the middle
of the atrioventricular canal before atrioventricular expansion
occurs51,52 or the atrioventricular canal expands, relative to
the muscular ventricular septum, to the right.41,53 The first
hypothesis was mainly based on an account of aggregating
trabeculae on the caudal wall of the left ventricle of a stage 12
(3.5 weeks) embryo.51,52 We (W.H.L., unpublished data,
1995) have reinspected the key specimen underscoring this
hypothesis and have interpreted the trabecular aggregation as
an artifact that was not seen in adjacent sections of the same
embryo or in other embryos of the same stage. The alternative
hypothesis requires a selective increase in the diameter of the
atrioventricular canal during atrioventricular septation. Although the diameter of the atrioventricular connection more
than doubles between 4.5 and 5.5 weeks of development
(14th and 15th stages), it remains constant between 5.5 and 7
weeks (16th through 19th stage),53 that is, during septation.
These data therefore strongly support our finding that the
right atrioventricular connection expands in the 7th week by
the formation of fenestrations in the floor of the tricuspid
gully.
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The “posterior (ie, caudal) smooth” or “sinus” septum on top
of the “trabeculated” muscular septum53 appears to refer to the
caudal or inlet component of the muscular ventricular septum
that we discussed earlier. In particular, its angular orientation
relative to the middle, trabecular portion of the muscular ventricular septum agrees with our observations. However, neither
its origin nor its cranial boundary, which we put at the bifurcation of the bundle branches, was delineated. The development of
the right-ventricular inlet from myocardium between the bundle
of His medially and the right bundle branch or septomarginal
trabeculation laterally has been hypothesized.54,55 The present
integration of the development of the atrioventricular canal with
that of the inlet portion of the ventricles into a single morphogenetic process has made it clear that malformations such as
double inlet left ventricle, atrioventricular septal defects, tricuspid atresia, and Ebstein’s anomaly should be considered as the
lasting consequences of a temporary arrest of normal
development.24,35,41,56
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Septation of the Outflow Tract
To understand septation in the outflow tract, a brief review of
its natural history is useful. The outflow tract connects the
embryonic right ventricle with the aortic sac and can first be
recognized in the 2nd half of the 4th week (12th stage).14 In
the 5th week of development (14th and 15th stages), the
outflow tract rapidly increases in length, while the ventricles
transform from peristaltoid to synchronously contracting
compartments14,57 (cf, Arguëllo et al58 and de Jong et al59).
The long sleeve of slowly conducting myocardium with
long-lasting contractions functions as a sphincter that prevents regurgitation during ventricular relaxation59 before the
appearance of the arterial valves in the 7th week. During this
period, the outflow tract can be divided into distinct proximal
and distal portions, separated by a distinct bend. During
contraction, the bend becomes accentuated, which may assist
in avoiding regurgitation. Only after its function as a sphincter and its phenotypic properties as primary myocardium
were revealed,59 the outflow tract became a developmental
entity, its proximal (upstream) and distal portion corresponding to the “conus” and “truncus,” respectively.60 Because
the terminology used to describe the respective parts of the
outflow tract continues to be contentious, we will use the
more descriptive terms proximal and distal portion.
The endocardial jelly within the outflow tract functions as
the stuffer material of the outflow-tract sphincter. In the
course of the 5th week, the cuff of the endocardial jelly
transforms into distinct septal and parietal ridges.14 This
transformation begins proximally and moves into the distal
outflow tract when the 6th arch arteries appear at 4.5 weeks
(14th stage).39 The septal ridge originates on the right side of
the trabeculated muscular ventricular septum just cranial to
the right bundle branch, whereas the parietal ridge originates
where the outflow tract meets the right ventricle and right
atrium in the inner curvature. Going from proximal to distal,
endocardial ridges follow a rightward spiraling course that
reflects, or at least follows, the rotational change in the
outflow tract that accompanies the process of looping, but
with a delay of 1 week. The sculpting of the ridges in the
distal outflow tract is accompanied by, and perhaps results
from, the penetration into them of neural crest– derived cells.
This invasion of neural crest cells may also trigger the onset
of fusion, from distal to proximal, of the ridges in the 6th
week.39,61 The fused ridges thus form a spiraling septum that
separates the outflow tract into a channel that connects to the
3rd and 4th arch arteries and a channel that connects to the 6th
arch arteries.
This description differs from the currently used model in
the role we attribute to the so-called aortopulmonary septum
in establishing septation in the distal outflow tract. The
prevailing view is that cells from the pharyngeal region
migrate toward the outflow tract to form its distal mesenchymal or truncal portion. In conjunction, neural crest– derived
mesenchyme is said to descend between the 4th and the 6th
branchial arches to divide, as the aortopulmonary septum, the
distal (mesenchymal or truncal) outflow tract into separate
aortic and pulmonary channels.62,63 The neural crest– derived
cells would further penetrate into the proximal outflow tract
as “prongs” of condensed mesenchyme within the septal and
parietal ridges.62– 64 However, this description condenses the
events that occur during the 5th and 6th weeks of development (14th through 17th stages) to such an extent that it
becomes inaccurate.
The increase in length of the outflow tract during the 5th
week is accompanied by distal expansion of the myocardium
along its outer layer, so that myocardium covers the entire
outflow tract up to its connection with the pharyngeal region
by the end of this week (15th stage57,65; J.-S. Kim, S. Webb,
A.F.M. Moorman, R.H. Anderson, W.H. Lamers, unpublished data, 2002). Similarly, the outflow tract in chicken
embryos increases in length between Hamburger and Hamilton stages 12 and 21.66 There is good reason to believe that
the (myocardial) cells forming this part of the outflow tract
are derived from the pharyngeal region in both mammals and
birds.67–70 This pharyngeal source of cardiomyocytes in the
outflow tract is now called the “anterior” or “secondary”
heart field.67,70 The phase of expansion of the myocardial
outflow tract is followed by a rapid regression of the
myocardium from its distal (truncal) portion toward the bend,
again both in mammals and in birds57,71 (Kim et al, unpublished data, 2002). The cardiomyocytes at the distal boundary
of the regressing myocardium show no signs of apoptosis,
suggesting that they transdifferentiate into the cells forming
the wall of the great arteries.57,71 This shortening of the
myocardial portion of the outflow tract in the 6th week, and
the coincident increase in the length of the nonmyocardial
arterial trunks therefore occurs after the pharyngeal cells of
the anterior heart field have contributed to the formation of
the outflow tract. The abrupt and absolute shortening of the
myocardial portion of the outflow tract has previously been
described as the “absorption of the conus.”66,72,73 Nevertheless, the distal myocardial boundary at the junction of the
outflow tract with the aortic sac as it is present at 5 weeks of
development is usually considered to be identical with the
position of the future arterial valves.62,72–75
The distal myocardial boundary of the outflow tract was
therefore implicitly considered to be a fixed landmark for the
position of the ventriculoarterial junction. Because the junction of the outflow tract with the 4th pair of branchial arches
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lies cranial to that with the 6th pair, whereas the root of the
aorta lies caudal to that of the pulmonary trunk, the apparent
change in position of the aortic and pulmonary roots has been
interpreted to result from rotation during septation.62,73 Such
a rotation of the ventriculoarterial junction would require a
compensatory downstream counterrotation. Instead, our analysis of outflow-tract development in rat and human embryos57 (Kim et al, unpublished data, 2002) shows that, concomitant with regression of the distal myocardial boundary, the
spiraling segments of the aortic and pulmonary channels
move from within to outside the myocardial heart. This
finding reveals that the intrapericardial portion of the aorta
and pulmonary trunk largely derives from the distal (truncal)
portion of the outflow tract and forms separate channels due
to fusion of the distal endocardial ridges. The observation that
the root of the great arteries contains hardly any neural crest
cells compared with the more distal parts61,76 underscores this
conclusion. The aortopulmonary septum, therefore, does not
descend from the aortic sac to the ventriculoarterial junction.
Instead, it is limited to a small, transversely oriented wedge of
tissue between the origins of the arteries supplying the 4th
and 6th branchial arches.
The downward migration of the neural crest cells of the
aortopulmonary septum is usually associated with formation
of the prongs of condensed mesenchyme within the septal and
parietal ridges.62– 64 In agreement with the near absence of
neural crest cells from the root of the aorta and pulmonary
trunks, we were unable to find a direct continuity between the
aortopulmonary septum and the prongs, suggesting that
the neural crest cells migrate through, but do not persist in the
distal outflow-tract ridges.76 At the 16th stage, the prongs
fuse and form the characteristic midline whorl just proximal
to the bend in the outflow tract57,62– 64 (Kim et al, unpublished
data, 2002). It is at this position that the valvar leaflets, still
surrounded by a cuff of outflow-tract myocardium, form from
the tissues of the endocardial ridges22,57,77 (Kim et al, unpublished data, 2002). The whorl and the proximal ends of the
prongs that connect the whorl to the lateral myocardial wall64
mark the plane of separation between the aortic and pulmonary
portions of the arterial valves. The aortic and pulmonary roots,
including the arterial sinuses, with their valvar leaflets, develop
in their entirety from the endocardial and intercalated ridges,
without a significant contribution from neural crest-derived
cells.57,61,76,78 In fact, the prominence of the neural crest– derived
cells during the period of fusion and their disappearance after the
ridges have fused76,79 suggest that their primary role is to assure
proper fusion of the ridges.80
The onset of formation of the valves corresponds with the
initiation of development of the coronary arteries. The myocardium that initially surrounds the arterial sinuses, including
the roots of the coronary arteries57 (Kim et al, unpublished
data, 2002), gradually disappears in the fetal period, the
sinutubular junction still marking the distal boundary of this
temporary myocardial cuff in the formed heart. The degenerate appearance of the myocytes just before their disappearance,57 coupled with the protracted course of the process,
suggests that regression of myocardium from the proximal
outflow tract is not a simple continuation of the initial
regression from the distal outflow tract. Incomplete regres-
Cardiac Septation
99
sion of this myocardium appears to be one of the causes of
ventricular tachycardia.80a While the myocardial cuff slowly
regresses, both arterial roots become separate structures that
begin to move apart in the 2nd trimester. The disappearance
of the myocardium that initially separates the developing left
coronary leaflet of the aortic valve and the medial leaflet of
the mitral valve is also part of this second wave of demyocardialization,57,81 but the degree to which this latter myocardial regression is completed varies.82 Nevertheless, the leftsided predominance of this regressive development is
striking, contrasting as it does with the increase in size of the
myocardium of the subpulmonary infundibulum.
The fusion of the proximal portion of the septal and
parietal ridges at 7.5 weeks (21st stage) completes the
septation of the outflow tract. At 6 weeks (17th stage), that is,
well before fusion, the portion of the outflow-tract ridges
below the arterial valves begins to myocardialize40,57,83,84
(Kim et al, unpublished data, 2002). The myocardialization
continues after fusion, producing a muscular partition between the subaortic and subpulmonary vestibules that does
not lie in line with the muscular ventricular septum. Instead,
the muscular partition is supported exclusively by the developing right ventricle, its proximal boundary (in the formed
heart) extending between the attachment of the medial papillary muscle on the ventricular septum and the lateral
boundary of the supraventricular crest at the junction of the
outflow tract with right ventricle and right atrium in the inner
curvature. As a result, the right ventricle and the subaortic
vestibule temporarily continue to communicate across the
muscular ventricular septum behind, that is, caudal to the
muscular partition. At this time, the atrioventricular cushions
have already fused (see earlier paragraph). The premier factor
that closes this remaining communication appears to be
growth of the muscular partition (Kim et al, unpublished data,
2002). If the communication persists, as is seen in many
hearts with ventricular septal defects, particularly tetralogy of
Fallot and double outlet right ventricle, the muscular partition
persists as an “outlet septum” between the subaortic and
subpulmonary vestibules. If the communication is closed, as
happens in normal development, it loses its septal position
(see later paragraph).
A key to understanding morphogenesis in the proximal
portion of the outflow tract is to appreciate its transformation
from a tubular structure at 5 weeks to a wedge-shaped
structure at 7 weeks of development. The asymmetric, rightsided development results from continued regression of
myocardium on the left side of the outflow tract (see above)
and continued growth in the corresponding right-sided part of
the outflow tract. Likewise, the muscular partition between
the subaortic and subpulmonary vestibules is cone-shaped
with its medial apex (derived from the septal ridge) to the
muscular ventricular septum and with its wide lateral base
(derived from the parietal ridge) to the lateral wall of the right
ventricle.
The muscular partition between the subaortic and subpulmonary vestibules is characterized by a distinctive parallel
orientation of its muscle fibers. Smooth muscle actin–positive
cells may play a prominent role in determining the parallel
course of the myocytes during their medial migration into the
100
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Figure 3. Development of the atrioventricular canal and ventricular inlets. Human embryos of 6 weeks (stage 17; A), 6.5 weeks (stage
18; B), 7 weeks (stage 19; C), and 9 weeks (D) were stained for the presence of ␤-myosin heavy chain, whereas human embryos of 5.5
weeks (stage 16), 6.5 weeks (stage 18), and 7 weeks (stage 19) were stained for the presence of CK-M. Hematoxylin-azoflochsin–
stained connexin40 and connexin43 double-deficient mouse fetuses35 at embryonic day 17 are shown in H though J. A through C,
Crest of the caudal portion of the muscular ventricular septum bends to the left (arrows) and appears to form a distinct part of the septum (D). This bent part of the septum supports the “tricuspid gully” (arrows in E through G). Participation of this part of the embryonic
right ventricle in septation is demonstrated by the varying degrees to which the inlet to the right ventricle has developed in the Cx40/
Cx43 doubly deficient mice (H through J). Hearts in H and I have a single atrioventricular junction. H, The most serious form, in which
the inlet to the right ventricle, including the tricuspid gully, is completely absent and resembles the heart at 5.5 weeks (D). I, Beginning
of the formation of the tricuspid gully and is comparable to the heart at 6.5 weeks (E). J, A heart with 2 unequal atrioventricular connections and a small right ventricular inlet and is comparable to a heart at 7 weeks of development (F). 1 indicates right appendage; 2,
primary atrial septum; 3, inferior endocardial cushion; 4, muscular ventricular septum; and 5, left ventricle.
muscular partition.57,85 The unique parallel alignment of the
myofibrils in the muscular partition remains identifiable in
the postnatal heart and permits its delineation in the caudomedial wall of the outlet portion of the right ventricle (Figure
4). The transition from a compact, cone-shaped muscular
partition to a long, relatively thin myocardial sheet that
connects the arterial valves of the aorta and pulmonary trunk
is a protracted event that begins at 8 weeks and is completed
only in the course of the second trimester. The flattening of
the muscular partition accentuates the offset between the
leaflets of the pulmonary and aortic valves. Because the
septal ridge is smallest and tethered to the muscular ventricular septum, the left-sided sinuses of the arterial valves,
which derive from this ridge, stay closest together, whereas
the right-sided sinuses, which derive from the bigger parietal
ridge, move furthest apart. Consequently, the attachment of
the right coronary cusp of the aortic valve projects on the
upstream end of the supraventricular crest, whereas the
corresponding cusp of the pulmonary valve forms the downstream boundary of the freestanding right-ventricular infundibulum. The muscular partition, therefore, continues to
connect the arterial valves of the aorta with those of the
pulmonary trunk, in doing so forming supraventricular crest
and the caudomedial wall of the freestanding right-ventricular
infundibulum. In the process, however, the muscular partition
loses its position as a ventricular outlet septum, which it had
in embryonic hearts.
The present account of outflow-tract development differs
in 2 aspects from existing descriptions. First, we have
explored the consequences of finding that the myocardium
that originates from so-called anterior heart67,68 regresses
after its function as the cuff of the outflow-tract sphincter
ceases57 (Kim et al, unpublished data, 2002). This led us to
appreciate that the proximal portion of the great arteries,
Lamers and Moorman
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Figure 4. Fate map of embryonic structures in the adult heart.
Indicators of orientation as used in this review are as follows:
Cr, cranial; Ca, caudal; D, dorsal; V, ventral; L, left; R, right.
Color codes identifying the embryonic origin of adult structures
are as follows: 1, sinus venosus; 2, primary atrial septum; 3,
secondary atrial septum; 4, atrial spine; 5, left/right appendage;
6, atrioventricular canal; 7, superior endocardial cushion; 8, inferior endocardial cushion; 9, ventricular inlet/tricuspid gully; 10,
left-lateral endocardial cushion; 11, outflow tract; 12, septal
endocardial ridge; and 13, parietal endocardial ridge/right-lateral
atrioventricular cushion. Adapted from Netter FH. Atlas of
Human Anatomy. Basel, Switzerland: Ciba-Geigy Ltd; 1989.92
including the sinuses the arterial valves, belong to the
embryonic outflow tract and, therefore, had a myocardial
external lining when first formed in the developing heart. The
bend between the proximal (conal) and distal (truncal) portions of the outflow tract can be traced to the sinutubular
junction in the great arterial trunks of postnatal hearts,
showing that the arterial valves develop from the distal
portion of the conus. Second, it was realized that the
myocardialization of the proximal (conal) portion of the
septum in the outflow tract should have important implications for the closure of the interventricular communication,
this being the last step in septation. In contrast to the
prevailing accounts of the final stages of septation (eg, van
Mierop87 and Los88), we attribute significance to the growth
of the muscular septum that separates the subaortic and
subpulmonary infundibula in completing septation. This explanation places the lack of sufficient growth or cranial
displacement, well-accepted explanations of the development
of double-outlet right ventricle and tetralogy, respectively,
into perspective as causes of septation defects. Finally, the
characteristic cytoarchitecture of the muscular septum allowed us to show how a septal structure in the outflow tract
becomes the myocardial caudomedial wall of the freestanding
right-ventricular infundibulum.
Our account shows that the muscular ventricular septum is
formed from 3 components: the primary or trabecular component that develops from the septum separating the trabeculated cavities of the developing left and right ventricles; the
inlet component that forms as part of the structures that bring
about septation of the atrioventricular junction; and the outlet
component that evolves from the structures that bring about
septation in the proximal outflow tract. The bifurcation of the
atrioventricular bundle into its bundle branches marks the
Cardiac Septation
101
boundary between the trabecular middle component and both
the inlet and the outlet components of the muscular ventricular septum. The bifurcation of the His bundle also marks the
divide between the contribution of the atrioventricular cushions and the outflow-tract ridges to septation, the atrioventricular cushions reaching to the bifurcation, and the endocardial ridges of the outflow tract starting distal to the right
bundle branch.
An important difference between the atrioventricular cushions and the outflow-tract ridges is that the former are made
up mostly of mesenchymal cells that are derived from the
endocardium,89 with the extracardiac mesenchymal contribution to atrioventricular septation remaining confined to the
atrial spine, whereas the outflow-tract ridges incorporate a
substantial contribution of neural crest– derived mesenchymal
cells.76,90,91 An additional, possibly associated, difference is
that the atrioventricular cushions do not become myocardialized, at least in man and in rodents, this process being
confined to the atrial spine. In contrast, the proximal portion
of the endocardial ridges of the outflow tract does become
myocardialized almost entirely to form the supraventricular
crest and the caudal wall of the freestanding right-ventricular
infundibulum (Kim et al, unpublished data, 2002).
Embryological Building Blocks of the
Formed Heart
A fate map of the formed heart may facilitate an active
understanding of septation. Fate maps obtained by following
physical markers are only available for avian embryos,5 but as
shown above, useful data can also be obtained by following
the distribution of phenotypical markers and the analysis of
congenital malformations. Figure 4 shows a fate map of the
human heart, mapping the structures described in this review.
Acknowledgments
This work was supported by the Department of Anatomy and
Embryology at the University of Amsterdam. The authors are
indebted to Drs R.H. Anderson, M.J.B. van den Hoff, and S. Webb
for critical reading of the manuscript and helpful suggestions for
improvement.
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Morphogenesis
Wouter H. Lamers and Antoon F.M. Moorman
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Circ Res. 2002;91:93-103
doi: 10.1161/01.RES.0000027135.63141.89
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